Bacteriophage transfer during faecal microbiota transplantation in Clostridium difficile infection is associated with treatment outcome

Abstract

Objective: Faecal microbiota transplantation (FMT) is effective for the treatment of recurrent Clostridium difficile infection (CDI). Studies have shown bacterial colonisation after FMT, but data on viral alterations in CDI are scarce. We investigated enteric virome alterations in CDI and the association between viral transfer and clinical outcome in patients with CDI.

Design: Ultra-deep metagenomic sequencing of virus-like particle preparations and bacterial 16S rRNA sequencing were performed on stool samples from 24 subjects with CDI and 20 healthy controls. We longitudinally assessed the virome and bacterial microbiome changes in nine CDI subjects treated with FMT and five treated with vancomycin. Enteric virome alterations were assessed in association with treatment response.

Results: Subjects with CDI demonstrated a significantly higher abundance of bacteriophage Caudovirales and a lower Caudovirales diversity, richness and evenness compared with healthy household controls. Significant correlations were observed between bacterial families Proteobacteria, Actinobacteria and Caudovirales taxa in CDI. FMT treatment resulted in a significant decrease in the abundance of Caudovirales in CDI. Cure after FMT was observed when donor-derived Caudovirales contigs occupied a larger fraction of the enteric virome in the recipients (p=0.024). In treatment responders, FMT was associated with alterations in the virome and the bacterial microbiome, while vancomycin treatment led to alterations in the bacterial community alone.

Conclusions: In a preliminary study, CDI is characterised by enteric virome dysbiosis. Treatment response in FMT was associated with a high colonisation level of donor-derived Caudovirales taxa in the recipient. Caudovirales bacteriophages may play a role in the efficacy of FMT in CDI.

Trial registration number: NCT02570477.

Keywords: BACTERIAL INFECTION; MUCOSAL INFECTION.

Figure 1

Virome alterations in Clostridium difficile infection (CDI). (A) Comparison of the relative abundance of enteric virus in CDI subjects and healthy controls at the order level. The bars indicate the median and IQR. Statistical significance was determined by Mann-Whitney test. *p<0.05. Comparison of Caudovirales diversity (B), richness (C) and evenness (D) between CDI subjects and healthy controls at the species level. Statistical significance was determined by Mann-Whitney test. *p<0.05. (E) Relative abundance of viruses at the family level. Statistical significance was determined by LEfSe analysis with FDR correction comparing all samples with all samples. * q<0.05. (F) Spearman correlation plots of Caudovirales, Microvirida and Anelloviridae in CDI subjects and controls. Statistical significance was determined for all pairwise comparisons; those with values of p< 0.05 are shown. Positive values (blue circles) indicate positive correlations, and negative values (red circles) indicate inverse correlations. The size and shading of the circle indicate the magnitude of the correlation, whereby darker shades are more correlated than lighter shades.

Figure 2

Alterations in the enteric virome after faecal microbiota transplantation (FMT). (A) Relative abundance of Caudovirales, Microviridae, and Anelloviridae in pre-FMT samples and post-FMT samples collected at the last follow-up. Statistical significance was determined by paired Wilcoxon sign permutation test, *p<0.05. (B) Alterations in the relative abundance of viral species in the stool of Clostridium difficile infection subjects after FMT at different time points until the last follow-up. ‘F’ indicates FMT-treated subject. ‘D’ indicates FMT donor. ‘W’ indicates weeks post treatment.

Figure 3

Alterations of Caudovirales diversity and richness after faecal microbiota transplantation (FMT). Changes in the Caudovirales diversity (A) and Caudovirales richness (B) of the stool samples of donor and Clostridium difficile infection (CDI) subjects after FMT at different time points until the last follow-up. (C) Presence–absence heat map of Caudovirales contigs in the stool samples of CDI subjects and their corresponding donors. Only contigs with reads per kilobase per million >1 were shown. ‘D’ indicates FMT donor; ‘R’ indicates FMT recipient. ‘Donor > recipient’ indicates the Caudovirales richness in stool samples of donor were higher than that of the recipient. ‘Responder’ indicates CDI subjects who responded to FMT; ‘non-responders’ indicates CDI subjects who had disease recurrence after FMT.

Figure 4

Transfer of Caudovirales bacteriophages and faecal microbiota transplantation (FMT) treatment outcome. (A) Presence–absence heat map of Caudovirales contigs in pre-FMT and post-FMT collected at the last follow-up for nine FMT recipients. Only contigs with reads per kilobase per million > 3 were shown to assure the colonisation of donor-derived contigs. Red lines indicate contigs transferred from the donor. (B) Percentage of donor-transferred Caudovirales contigs in FMT recipients at the last follow-up. The size of the circle indicates the count of Caudovirales contigs transferred from donor. The colour of the circle indicates the richness of Caudovirales in the recipient relating to the treatment response. (C) Comparison of the frequency of donor-derived Caudovirales contigs in FMT responders and in non-responders. Statistical significance was determined by Mann-Whitney test. *p<0.05. (D) Presence of bacterial operational taxonomic unit (OTUs) in FMT recipients at the last follow-up. The colour of the bar indicates the origin of the bacterial OTUs. Purple indicates donor-derived OTUs colonised in the recipient, orange indicates OTUs exclusively present in recipient but not in the donor, while green indicates OTUs present both in donor and in recipient. (E) Comparison of the frequency of donor-derived bacterial OTUs in FMT responders and in non-responders. Statistical significance was determined by Mann-Whitney test.

Figure 5

Bacteria–Caudovirales relationship pattern (A) The correlation of bacteria richness with Caudovirales diversity and bacteria diversity with Caudovirales diversity in Clostridium difficile infection (CDI) and controls, respectively. Linear regression±95% CI, Spearman correlation coefficient and p value are shown. (B) The correlation of bacteria richness with Caudovirales diversity and bacteria diversity with Caudovirales diversity before and after faecal microbiota transplantation (FMT). (C) Bacteria–Caudovirales correlation pattern during FMT treatment. Spearman correlation plots of the relative abundances of Caudovirales species and bacterial families identified to be significantly associated with CDI and controls, in donor, pre-FMT and post-FMT samples. Statistical significance was determined for all pairwise comparisons; significant correlations (p<0.05) are displayed with asterisk. Blue circles and positive values indicate positive correlations, and red circles and negative values indicate inverse correlations. The size and shading indicate the magnitude of the correlation, where darker shades are more correlated than lighter ones.